Catalysts are utilized in 80% of all chemical synthesis operations. The industrial catalysts primarily used in oxidation reactions are highly polluting and expensive metal catalysts. Enzymes and whole cell biocatalysts are used to a lesser extent. Nowadays, several industrial sectors are developing bio-based technologies to reduce the high costs and environmental impact of traditional chemical processes. However, these applications are limited by the challenge of developing economically competitive biologically based systems. The key for adopting these sustainable advancements is the development of novel process designs, which assure robustness, simplicity, and sustainabile operations compatible with the current development of chemical reactions. In this regard, filamentous fungi may be considered good biocatalysts due to their natural biodiversity and their broad heterogeneous enzymatic pattern. The great selectivity of fungal catalysis is now well recognized for the production of commercially valuable steroids in the pharmaceutical industry. Although this inherent capacity is mainly used for functionalization of unactivated carbons, it can be further exploited for the oxidiation of heteroatoms, such as sulfur. Focusing on the oxidation of sulfur compounds, the widely used industrial processes are produced by an organometallic catalyst. This PhD project aims to overcome low substrate conversion and enzymatic expression by proving that exposure of cells to insecticides and hydrocarbons increases cell's oxidative capacity expressed as higher substrate conversion and CYP450 content. This study is focused in the application of pest management strategies, designed to enhance the biopesticide's efficacy, to induce and improve Beauveria bassiana oxidation. B. bassiana has a very flexible metabolism and is widely used as a biocontrol agent. It can metabolize hexadecane as a sole carbon source. In addition, it shows a synergistic effect over pest control efficacy when it is applied with low pesticides (carbaryl and/or imidacloprid) concentrations. A biocatalytic system was optimized to increase the conversion of organosulfur compounds under different fermentation conditions. Phenothiazine was used as our model substrate. Phenothiazine conversion was followed by GC-MS and HPLC. By NMR and MS fragmentation pattern product, phenothiazine metabolites were identified as (R)-hydroxyl metabolites (63% enatiomeric excess) and sulfoxide, the latter being the main metabolite. Phenothiazine conversions with growing cells resulted in 65±1.4% conversion with initial phenothiazine concentration of 500 ppm and final 325 ppm after 7 days. The highest conversion, 74±1 % was achieved with resting cells at the lowest cell concentration, 0.78 mg cell dry weight (cdw) /mL. Furthermore, the use of insecticides as inducers was an effective way to increase phenothiazine conversion from 47% to 64±3%. The major enzymes involved in catalysis of xenobiotic are heme-binding monooxygenases, in particular cytochrome P450. Heme positive proteins were identified by an SDS benzidine assay as well as the content of CYP450 by the CO difference spectrum. The P450 enzymes content was 12.3±1 pmol/µg protein for hexadecane adapted cells and 8.1± 1 pmol/µg protein for insecticides, respectively. The heme-positive proteins were characterized by MALDI-ToF and their peptide mass fingerprint compared to the available sequences on the SwissProt/Universal Protein Resource catalog of information on proteins (UniProtKB). Hemoproteins were found, including a cluster of catalase-peroxidase, alkane hydroxylase, and chloroperoxidase. The results from this project helped bridge the progress from agricultural biotechnology strain development into industrial biotechnology biocatalyst improvement. The success of this project helps us expand B. bassiana's catalysis and make it a better candidate for industrial biocatalysis.

No description available.

Pathogenic fungi, such as polypore fungi that infect live sapwood, decrease quality and value of wood; however their effects on canopy physiology and growth have been little examined. This study examines how Fomes fomentarius, a species of polypore fungus affects canopy physiology in Betula alleghaniensis. A mobile canopy lift enabled the collection of leaf physiology, morphology and chemistry data from canopies of infected, damaged, and control trees. A molecular protocol developed to detect and identify polypore fungi in live trees confirmed that F. fomentarius was the major species present in infected trees. Infected trees exhibited reductions in physiological performance and growth, along with higher leaf carbon and chlorosis. While some characteristics of fungal infection were consistent with a mechanism involving partial xylem occlusion, patterns did not resemble those of a simple drought response. Likely, other factors such as fungal toxins or host defense mechanisms also contribute to these patterns.

Pathogenic fungi, such as polypore fungi that infect live sapwood, decrease quality and value of wood; however their effects on canopy physiology and growth have been little examined. This study examines how Fomes fomentarius, a species of polypore fungus affects canopy physiology in Betula alleghaniensis. A mobile canopy lift enabled the collection of leaf physiology, morphology and chemistry data from canopies of infected, damaged, and control trees. A molecular protocol developed to detect and identify polypore fungi in live trees confirmed that F. fomentarius was the major species present in infected trees. Infected trees exhibited reductions in physiological performance and growth, along with higher leaf carbon and chlorosis. While some characteristics of fungal infection were consistent with a mechanism involving partial xylem occlusion, patterns did not resemble those of a simple drought response. Likely, other factors such as fungal toxins or host defense mechanisms also contribute to these patterns.

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Made available in DSpace on 2014-06-11T19:32:53Z (GMT). No. of bitstreams: 0 Previous issue date: 2013-03-01Bitstream added on 2014-06-13T21:05:08Z : No. of bitstreams: 1 voltan_ar_dr_arafcf.pdf: 1013360 bytes, checksum: 7a427a197309b3a1913694635297ee6e (MD5) Bitstreams deleted on 2015-05-25T13:05:07Z: voltan_ar_dr_arafcf.pdf,. Added 1 bitstream(s) on 2015-05-25T13:05:43Z : No. of bitstreams: 1 000715398_20160301.pdf: 174801 bytes, checksum: 8a2d22fd4d7938bc5ecd9d9d0fdd4b6c (MD5) Bitstreams deleted on 2016-03-02T17:37:22Z: 000715398_20160301.pdf,. Added 1 bitstream(s) on 2016-03-02T17:38:08Z : No. of bitstreams: 1 000715398.pdf: 978625 bytes, checksum: f9f26554857da3d03570f04ef587630d (MD5) / Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) / Os fungos dimórficos, Paracoccidioides brasiliensis (espécies cripticas S1, PS2, PS3) e Paracoccidioides lutzii (Pb01-like espécies), são agentes da paracoccidioidomicose (PCM), doença granulomatosa crônica, endêmica na América Latina, principalmente no Brasil. A doença apresenta grande variedade de manifestações clínicas, desde formas localizadas até disseminadas evoluindo para letalidade. O fungo tem capacidade de aderir, invadir e extravasar barreiras impostas pelos tecidos do hospedeiro. P. brasiliensis (Pb18) já foi observado tanto no interior de macrófagos, como no interior de células epiteliais in vivo e in vitro. A identificação do mecanismo pelo qual este fungo sobrevive no interior da célula hospedeira é campo fértil para a descoberta de sua patogênese, já que este microrganismo possui a capacidade de induzir sua própria endocitose em células epiteliais e muito provavelmente em macrófagos. A absorção de micronutrientes pelo fungo apresenta papel singular, tanto para sua nutrição e processo invasivo, como para sua sobrevivência no interior da célula hospedeira. A via endocítica em microrganismos é de fundamental importância na regulação de todo esse processo. O objetivo deste estudo foi avaliar a via endocítica de Pb18 e também de macrófagos infectados com este fungo. A avaliação da via endocítica de Pb18 foi realizada na condição de depleção de metais, onde foram analisados os genes Clatrina e Ypt7 (Homólogo de Rab7) por RT-PCR semi-quantitativo e PCR em tempo real (RT-PCR quantitativo). Também foi analisada a infecção de macrófagos por Pb18 cultivado na depleção de metais e em diferentes tensões de oxigênio. A via endocítica de macrófagos foi analisada por RT-PCR quantitativo e imunofluorescência. Esta análise foi realizada quando o fungo foi cultivado em... / The dimorphic fungus, Paracoccidioides brasiliensis (cryptic species S1, PS2, PS3) and Paracoccidioides lutzii (Pb01-like species), are agents of paracoccidioidomycosis (PCM), chronic granulomatous disease, endemic in Latin America, especially in Brazil. The disease has a wide variety of clinical manifestations, from localized forms to disseminated evolving to lethality. The fungus is able to adhere, invade and spill barriers imposed by host tissues. P. brasiliensis (Pb18) has already been observed both within macrophages, but also inside of epithelial cells in vivo and in vitro. Identification of the mechanism by which this fungus survive within the host cell is a fertile field for the discovery of their pathogenesis, since this microorganism has the ability to induce its own endocytosis in epithelial cells and most probably in macrophages. The micronutrient uptake by the fungus presents unique role, both for its nutritional and invasive procedure, such as for survival within the host cell. The endocytic pathway in microorganisms is of fundamental importance in the regulation of this process. The aim of this study was to evaluate the endocytic pathway of Pb18 and also macrophages infected with this fungus. The evaluation of the endocytic pathway of Pb18 was performed under the condition that depletion of metals, where the genes were analyzed Clathrin and Ypt7 (Rab7 homolog) by RT-PCR semi-quantitative and real-time PCR (RT-PCR quantitative). Was also analyzed by Pb18 infection of macrophages cultured in the depletion of metals at different oxygen tensions. The endocytic pathway of macrophages was analyzed by quantitative RT-PCR and immunofluorescence. This analysis was performed when the fungus was grown at different oxygen tensions and macrophages were infected with this and when the infection was incubated at... (Complete abstract click electronic access below)

Orientador: Maria José Soares Mendes Giannini / Coorientador: Ana Marisa Fusco Almedia / Banca: Gil Benard / Banca: Marcelo Pelajo Machado / Banca: Christiane Pienna Soares / Banca: Raquel Mantuaneli Scarel Caminaga / Resumo: Os fungos dimórficos, Paracoccidioides brasiliensis (espécies cripticas S1, PS2, PS3) e Paracoccidioides lutzii (Pb01-like espécies), são agentes da paracoccidioidomicose (PCM), doença granulomatosa crônica, endêmica na América Latina, principalmente no Brasil. A doença apresenta grande variedade de manifestações clínicas, desde formas localizadas até disseminadas evoluindo para letalidade. O fungo tem capacidade de aderir, invadir e extravasar barreiras impostas pelos tecidos do hospedeiro. P. brasiliensis (Pb18) já foi observado tanto no interior de macrófagos, como no interior de células epiteliais in vivo e in vitro. A identificação do mecanismo pelo qual este fungo sobrevive no interior da célula hospedeira é campo fértil para a descoberta de sua patogênese, já que este microrganismo possui a capacidade de induzir sua própria endocitose em células epiteliais e muito provavelmente em macrófagos. A absorção de micronutrientes pelo fungo apresenta papel singular, tanto para sua nutrição e processo invasivo, como para sua sobrevivência no interior da célula hospedeira. A via endocítica em microrganismos é de fundamental importância na regulação de todo esse processo. O objetivo deste estudo foi avaliar a via endocítica de Pb18 e também de macrófagos infectados com este fungo. A avaliação da via endocítica de Pb18 foi realizada na condição de depleção de metais, onde foram analisados os genes Clatrina e Ypt7 (Homólogo de Rab7) por RT-PCR semi-quantitativo e PCR em tempo real (RT-PCR quantitativo). Também foi analisada a infecção de macrófagos por Pb18 cultivado na depleção de metais e em diferentes tensões de oxigênio. A via endocítica de macrófagos foi analisada por RT-PCR quantitativo e imunofluorescência. Esta análise foi realizada quando o fungo foi cultivado em... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: The dimorphic fungus, Paracoccidioides brasiliensis (cryptic species S1, PS2, PS3) and Paracoccidioides lutzii (Pb01-like species), are agents of paracoccidioidomycosis (PCM), chronic granulomatous disease, endemic in Latin America, especially in Brazil. The disease has a wide variety of clinical manifestations, from localized forms to disseminated evolving to lethality. The fungus is able to adhere, invade and spill barriers imposed by host tissues. P. brasiliensis (Pb18) has already been observed both within macrophages, but also inside of epithelial cells in vivo and in vitro. Identification of the mechanism by which this fungus survive within the host cell is a fertile field for the discovery of their pathogenesis, since this microorganism has the ability to induce its own endocytosis in epithelial cells and most probably in macrophages. The micronutrient uptake by the fungus presents unique role, both for its nutritional and invasive procedure, such as for survival within the host cell. The endocytic pathway in microorganisms is of fundamental importance in the regulation of this process. The aim of this study was to evaluate the endocytic pathway of Pb18 and also macrophages infected with this fungus. The evaluation of the endocytic pathway of Pb18 was performed under the condition that depletion of metals, where the genes were analyzed Clathrin and Ypt7 (Rab7 homolog) by RT-PCR semi-quantitative and real-time PCR (RT-PCR quantitative). Was also analyzed by Pb18 infection of macrophages cultured in the depletion of metals at different oxygen tensions. The endocytic pathway of macrophages was analyzed by quantitative RT-PCR and immunofluorescence. This analysis was performed when the fungus was grown at different oxygen tensions and macrophages were infected with this and when the infection was incubated at... (Complete abstract click electronic access below) / Doutor

Nas últimas décadas, houve aumento progressivo das infecções hospitalares por leveduras principalmente, do gênero Candida. A fonte de infecção pode ser endógena ou exógena, desde que esporos unicelulares de leveduras, permanecem viáveis por meses sobre superfícies bióticas ou abióticas. Diversas espécies de leveduras são encontradas em pele e mucosas de indivíduos sadios. Em estado saprofítico ainda, as leveduras encontram-se no trato gastrintestinal humano mas, a relação entre a presença desses microorganismos e sua patogenicidade está associada a diversos fatores predisponentes, tais como: número e variedade de sítios topográficos colonizados, uso prolongado de antibióticos, infecções associadas causadas por outros microorganismos e particularmente, distúrbios imunológicos ou metabólicos. Leveduras do trato gastrintestinal podem ser transmitidas, via fecal-oral diretamente ou de modo indireto, de indivíduo para indivíduo. A transmissão de uma cepa em estado saprofítico para um hospedeiro suscetível, pode resultar em colonização seguida de infecção. A gravidade do quadro clínico depende de condições do hospedeiro e características do agente etiológico que engloba fatores de virulência e resistência a antifúngicos. Esses atributos são importantes em Candida albicans na qual, enzimas com atividade de fosfolipase e proteinase são marcadores de virulência. De outro modo, fenótipos de resistência, ocorrem, com maior freqüência, em espécies não-Candida albicans. Dada a possibilidade de instalação de doença endógena e a dispersão de cepas virulentas e resistentes, a partir de colonização gastrintestinal, estudos que contribuam para a determinação desses agentes constituintes da microbiota de pacientes internados, são importantes para o conhecimento da história natural das infecções nosocomiais por leveduras. Os objetivos deste trabalho foram avaliar o trato intestinal como fonte potencial de infecção hospitalar por leveduras, descrevendo as espécies prevalentes nas primeiras horas de internação e possíveis alterações temporais, quanto a fenótipo de virulência e resistência a antifúngicos. Foram analisadas 281 amostras de leveduras isoladas de 66 crianças internadas em unidades de pediatria e semi-intensiva de hospitais públicos das cidades de São Paulo e Guarulhos, Brasil. As amostras foram isoladas de fezes coletadas nas primeiras horas de internação e durante o período de internação. A identificação das leveduras quanto a gênero e espécie foram realizadas por métodos tradicionais, analisando aspectos morfológicos e fisiológicos. A capacidade de produção de enzimas, fosfolipase e proteinase, foram verificadas conforme proposto por Price et al. 1982 e Ruchël et al., 1982. A sensibilidade aos antifúngicos: anfotericina B (AMB), fluconazol (FZ), itraconazol (IZ), cetoconazol (CZ) e nistatina (NIS) foram analisada pela técnica de difusão por discos (CECON São Paulo, Brasil). Amostras resistentes ou com sensibilidade intermediária, foram re-avaliadas pelo método de microdiluição segundo NCCL (1997) modificado por EUCAST (2002). As espécies isoladas foram: Candida tropicalis (32,7%), C.albicans (29,9%), C.parapsilosis (27, 1%), Trichosporon cutaneum e T.inkin (3,2%), Rhodotorula mucilaginosa e R.glutinis (0,7%), C.krusei (3,6%), C.guilliermondii (2,1%), C.glabrata (0,4%) e C.kefyr (0,4%). A atividade enzimática foi observada na maioria das 84 amostras de C. albicans, sendo 96% de fosfolipase e 95% de proteinase. Entre as espécies não-albicans do gênero Candida foi verificada atividade em 97% de fosfolipase e 67% de proteinase. Amostras menos sensíveis às drogas azólicas, ou seja, amostras resistentes ou com sensibilidade dependente da dose, foram encontradas em 4,3% das 281 amostras de leveduras, sendo maior porcentagem observada em C.krusei (90%). Conclui-se que existem leveduras de diversas espécies em fezes de população pediátrica hospitalizada, com fenótipos de virulência e resistência a antifúngicos. A manutenção desses fenótipos durante o período de internação pode representar fator de risco para infecção hospitalar endógena, ou ainda, fonte de dispersão de patógenos em potencial, no meio ambiente hospitalar. / At the last decades the nosocomial infections caused by yeasts raised significantly especially by Candida yeasts. The infections source can be endogen or exogenous, since spores of unicellular and multicellular are kept viable for months and several yeasts species are found in skin and mucosa of healthy people. In a saprophytic state yeasts are found in the human gastrointestinal tract but the relationship between the presence of these microorganisms and their pathology is associated with several facts such as: number, variety of sites colonized, effective use of antibiotics, associated infections caused by another microorganisms and mainly disturbance in due to lack of immunity and metabolic. Yeasts in the gastrointestinal tract can be transmitted fecal-oral direct or indirectly from an individual to another. The transmission of a strain in a saprophytic state to a host can result in colony followed by infection. The infection can be serious depending on the host conditions and the etiologic agent that includes virulent factor and resistance to antifungal drugs. These attributes are important to Candida albicans in which enzymes with phospholipase activity are responsible for virulent factors. Resistance phenotypes, otherwise it should occur more frequently in non-albicans species. Concerning the possibility of an endogen disease and the spread of virulent and resistant strains, from the gastrointestinal colony, studies that contribute to determine these agents that constitute the microbiota of patients, are important to know the natural story of nosocomial infections caused by yeasts. This work aims at evaluating the intestinal tract as a source of hospital infections by yeasts describing the remaining species in the first hours and a possible change depending on the time that may happen to virulent phenotypic and resistance to ant fungi. Two hundred eighty one yeast samples from sixty-six children attended in pediatric and semi-intensive units in 2 public hospitals located in São Paulo and Guarulhos cities in Brazil were analyzed. The fecal samples were collected at the first hours after and during their arrival at the hospital. To identify the yeasts according to their gender and species traditional methods were used, analyzing morphological and physiological aspects. The ability to produce enzymes phospholipase and proteinase was verified the same way it was proposed by Price et al. 1982 and Ruchel et al. 1982. The sensibility to antifungals: amphotericin B (AMB), f1uconazole (FZ), ketoconazole (CZ) e nistatin (NIS), was analyzed by the diffusion technical by disks (CECON São Paulo, Brazil). Resistant samples or with intermediate sensibility were confirmed by micro-dilution method according to NCCLS (1997) modified by EUCAST (2002). The isolated species were: Candida tropicalis (30%), C.parapsilosis (27%), C.krusei (4%), Trichosporon cutaneum e T.inkin (3%), Rhodotorula mucilaginosa e R.glutinis (2%), C.guilliermondii (2%), C.glabrata (1%) and C.kefyr (1%). Enzymatic activity was verified in most of the 84 C.albicans samples being 96% of phosfolipase and 95% of proteinase production. Among the non-albicans species of Candida it was observed 97% of phospholipase and 67% of proteinase activity. Less sensitive samples to azoic drugs including resistant or SDD sensibility, which depends on the achieved dose, were found in 4.3% of the 281 samples of yeast. The hugest percentage was observed in C.krusei (90%). We can conclude that different yeast species occur in stools of pediatric population hospitalized, including virulent strains and antifungal resistant phenotypes. The persistent of these phenotypes in the intestinal tract during hospitalization period may represents a risk facto r contributing to endogen infection, or play a role in dissemination of potential pathogens inside a nosocomial environment.

Orientador: Diego Cunha Zied / Resumo: O fungo Lecanicillium fungicola é um importante patógeno no cultivo de Agaricus bisporus, apresentando diversos sintomas e perdas de produção. O objetivo do trabalho foi avaliar interações in vivo e in vitro dos fungos Lecanicillium fungicola e Agaricus bisporus. Os experimentos in vitro foram feitos utilizando discos de micélio do patógeno e composto colonizado pelo A. bisporus. O composto colonizado também foi utilizado para a frutificação de cogumelos tanto em tamanho menor em placas de petri como em maior quantidade. Foram aplicados quatro suspensões de esporos L. fungicola nos cogumelos em placas de petri, de acordo com o seu tratamento. Não ocorreu a paralisação do crescimento micelial in vitro no momento em que o patógeno e o hospedeiro se encontram. Após 36 horas a inoculação do patógeno foi possível a visualização de manchas no píleo e após 60 horas a visualização de hifas e esporos. A linhagem coloração creme apresentou maior massa e diâmetro do píleo e menor porcentagem de rompimento do véu. A linhagem de A. bisporus de coloração branca obteve maior produtividade do que a linhagem de coloração creme. O isolado LF 19/03 apresentou maior agressividade em ambas as linhagens de A. bisporus. Os diversos sintomas encontrados no decorrer da pesquisa possibilitaram a confecção de uma escala diagramática para auxiliar o produtor na comercialização dos cogumelos doentes. / Abstract: The fungus Lecanicillium fungicola is an important pathogen in the cultivation of Agaricus bisporus, presenting several symptoms and production losses. The objective of the work was to evaluate interactions in vivo and in vitro of the fungi Lecanicillium fungicola and Agaricus bisporus. The in vitro experiments were done using mycelium discs of the pathogen and compound colonized by A. bisporus. The colonized compost was also used for the fruiting of mushrooms both in smaller size in petri dishes and in greater quantity. Four suspensions of L. fungicola spores were applied to the boxes according to their treatment. Mycelial growth did not stop in vitro at the time the pathogen and host meet. After 36 hours the inoculation of the pathogen made it possible to see spots on the cap and after 60 hours, to view hyphae and spores. The cream-colored lineage showed greater mass and diameter of the cap and lesser percentage of rupture of the veil. The white colored A. bisporus strain obtained higher productivity than the cream colored strain. The isolate LF 19/03 showed greater aggressiveness in both strains of A. bisporus. The various symptoms found in the course of the research enabled the production of a diagrammatic scale to assist the producer in the sale of sick mushrooms. / Mestre

<p>Over the last two decades, mycoflorostic studies undertaken in Guyanese forests have uncovered hundreds of new fungal species and genera. One of the recovered fungal families was the Xylariaceae, although most were not identified to species. Members of this family play ecological roles as decomposers, endophytes, and pathogens of vascular plants and grass species. In addition, this group is increasingly recognized as a significant source of novel metabolic products with potential for applications in medicine, agriculture, and industrial biofuel. Given its potential, we took a more targeted approach to the documentation of this group. Our sampling efforts drawing on more than two decades of field collections yielded ca. 90 species in 12 genera, including a putative pathogen, <em>Xylaria karyophthora</em> of the seeds of <em>Chlorocardium</em> spp (Greenheart). Despite the significance of Greenheart to the Guyanese economy, little is known about the biology and ecology of this fungus. Due to the lack of available resources to study this fungus, our objectives were two-fold: first, to sequence and annotate the genome of <em>X. karyophthora</em> to provide a resource for genome-centric explorations, and to use this genome to infer the biology and ecology of this fungus. We focused on identifying and characterizing secretomes, viz. carbohydrate-active enzymes (CAZymes) and secondary metabolites biosynthetic gene clusters (SMBGCs) to infer the nutritional strategy of this fungus. Our results suggest that <em>X. karyophthora</em> has the capacity to act as both an endophyte and a pathogen. To make further inferences about the population, we used SSR markers to elucidate the genetic diversity and population structure of <em>X. karyophthora</em>. <em>X. karyophthora</em> populations have high genetic diversity, potentially exploiting both outcrossing and inbreeding reproductive strategies, and demonstrate a pattern consistent with human-mediated spread. This work will contribute information on new species and records of Xylariaceous fungi and their allies from Guyana with particular emphasis on unraveling the epidemiology, genetic diversity, and population structure of <em>X. karyophthora.</em></p>